Structural and functional insights into the p160 Rho-associated coiled-coil-containing protein kinase ROCK

Rho-associated protein kinases (ROCK1/2) play a key role in many fundamental cellular processes and are promising therapeutic targets for various diseases, particularly cardiovascular disease. However, unlike the terminal globular kinase domain and the PH-C1 tandem (PHn-C1-PHc), the sequence-structure-function relationship of the central amphipathic α-helical segment of ROCK proteins is not well understood. This study employed multiple biophysical techniques, including multi-angle light scattering (MALS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), negative-stain electron microscopy (EM), X-ray crystallography, and 3D molecular modeling to elucidate the structural basis of the entire ROCK1 segment. The study yielded significant new insights into the structural organization of ROCK proteins. Notably, the crystal structure of the homology region 1 (HR1) of ROCK1 (ROCK ^HR1 ), located at the N-terminus of the amphipathic segment and determined at 2.2 Å resolution, provided the first experimental evidence of a canonical parallel coiled-coil dimer. This structure differs significantly from the antiparallel coiled-coil HR1 module found in other Rho effector kinases. Along with the structural reconstitution of full-length ROCK1, this evidence demonstrates that the central segment of ROCK1 forms a highly elongated coiled-coil dimer. EM analysis using purified ROCK ^FCC revealed that this dimer switches between a dimer and a tetramer.